Spinal Muscular Atrophy (SMA) is one of the most common lethal genetic diseases in children. One in thirty five people carry a mutation in a gene called survival of motor neurons 1 (SMN1) which is responsible for this disease. If two carriers have children together they have a one in four chance of having a child with SMA. Children with Type I SMA seem fine until around 6 months of age, at which time they begin to show lack of muscular development and slowly develop a "floppy" syndrome over the next 6 months. Following this period, SMA children become less able to move and are eventually paralyzed by the disease by 3 years of age or earlier. We know that this mutation causes the death of motor neurons - which are important for making muscle cells work. Interestingly, there is a second gene which can lessen the severity of the disease process (SMN2). Children with more copies of this modifying gene have less severe symptoms and can live for longer periods of time (designated Type II, III and IV and living longer periods respectively).
There is no therapy for SMA at the current time. One of the roadblocks is that there are no human models for this disorder as it is very difficult to make the motor neurons that die in the disease in the laboratory. The researchers in the current proposal have recently created pluripotent stem cells from a patient with Type I SMA (the most severe) and shown that motor neurons grown out from the pluripotent stem cells also die in the culture dish just like they do in children. This is an important model for SMA.
The proposed research takes this model of SMA and extends it to Type II and Type III children in order to have a wider range of disease severity in the culture dish (Type IV is very rare and difficult to get samples from). It then develops new technologies to produce very large numbers of motor neurons and perform large scale analysis of their survival profiles. Finally, it will explore whether novel compounds can slow down the degeneration of motor neurons in this model which should lead to the discovery of dew drugs that then may be used to treat the disease.

Statement of Benefit to California:

The aim of this research is to develop novel drugs to treat a lethal childhood disease - SMA. There would be three immediate benefits to the state of California and its citizens.
1. Children in California would have access to novel drugs to slow or prevent their disease.
2. SMA is a world wide disease. The institutions involved with the research would be able to generate income from any new drugs developed and the profit from this would come back to California.
3. The project will employ a number of research staff in Californian institutions

Progress Report:

Year 1

This year we have created a large number of new SMA lines, developed ways to differentiate them into motor neurons using high content dishes, and begun to analyze the health of the motor neurons over time. We have also submitted a new paper showing that much of the cell death seen in the dying motor neurons is due to apoptosis - a form of cell death that is treatable with specific types of drug. We are now using these new lines to begin setting up screening runs with drug libraries and should be able to start these in the new year of funding.

Year 2

In this year we have made more induced pluripotent stem (iPSC) cell lines from Spinal Muscular Atrophy patients also using blood cells in addition to skin cells. Blood cells from patients are usually more readi;y accessible. As such, this technique can be used to make larger bank of similar cell lines. We have also rigorously tested all the iPSCs them for their quality. These lines are now available for distribution to other California researchers along with a certificate of analysis.
Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in large quantities of millions to billions in number. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients. To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMNPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have made these iMNPS now from many SMA patients as well as healthy humans. These spheres can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons.
Deficiency of an important protein in SMA patients is one of the key causes to the course of the disease. We have also designed an automated method for identifying new drugs in patient motor neurons that will test for correction of SMN protein levels in motor neurons.

Year 3

In Year 3 we completed making all iPSC lines from Spinal Muscular Atrophy patients. We rigorously tested all the iPSCs for quality. These lines are now available for distribution to other California researchers along with a quality control certificate.
Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in billions and repeatedly. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients.
To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have now tested our method in multiple patient cells and characterized these spheres. The iMPS have now been produced from many SMA patients as well as healthy humans. The next step we have developed is to take the iMPS to make motor neurons that are similar to those that are affected in SMA children. We have then discovered a method for creating them quickly. These aggregate spheres and spinal cord motor neurons from them can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons. Using new technologies that can edit, cut, copy, and paste new DNA in the stem cell genome, we are also developing ways to engineer iPS cell lines that will tag the motor neurons when they are made. This will allow us another method for making pure motor neurons and tracking them in a dish among other types of cells while they are alive.
Deficiency of an important SMN protein in SMA patients is one of the key causes to the course of the disease. An automated method has been developed for identifying what causes the SMA neurons to become sick and test new drugs in motor neurons. We are now gearing up to test some ~1400 known compounds on patient motor neurons to determine whether we can raise SMN protein levels in motor neurons.

Year 4

The goal of this project has now been reached. We have developed a new screening platform using motor neurons from induced pluripotent stem cells taken from children with spinal muscular atrophy. Through this technology we have screened thousands of compounds and have shown a small sub set that active gene expression and enhance motor neuron survival in this model. These compounds will now be moved to the next stage for validation. This funding has allowed us to complete the development of this tool/technology and put us in a strong position to continue these studies and the drug development process to move interesting drugs to the market for spinal muscular atrophy.